Background/Objectives: Carvedilol is an adrenergic blocker FDA-approved to improve outcomes in heart failure with reduced ejection fraction. Clinical trials examining whether carvedilol may be cardioprotective in the setting of cancer therapy-induced heart failure have generated mixed results that may depend on the cancer regimen, tumor, or comorbidities. Methods: To investigate the therapeutic potential of carvedilol to mitigate doxorubicin cardiotoxicity in cardiomyocytes, myocardial tissue, and in vivo, independent of confounding factors in clinical studies, we utilized disease-free cardiac slices and cardiomyocytes from mice, dogs, and human in vitro, and in wildtype mice injected with doxorubicin in vivo. We further evaluated the impact of carvedilol in dogs with cancer receiving doxorubicin. Results: In primary canine and murine cardiac slices, carvedilol treatment restored autophagy and prevented apoptosis from doxorubicin. Carvedilol restored mitochondrial energetics in human, canine, and murine models. In wildtype mice challenged with doxorubicin, carvedilol prevented declines in cardiac function and alterations in cardiac structure. In pet dogs with cancer and undergoing doxorubicin treatment, carvedilol was beneficial in preserving cardiac function and structure. Conclusions: Carvedilol activates cardioprotective autophagy, arrests doxorubicin-induced cell death, and improves energetics and cardiac structure and function across species.

Autophagy, an evolutionarily conserved process, plays an important role in cellular homeostasis and human diseases. Cardiovascular dysfunction, which presents during cancer treatment or in cancer-free individuals years after treatment, is a growing clinical challenge. Millions of cancer survivors and patients face an unpredictable risk of developing cardiotoxicity. Cardiotoxicity due to cancer treatment, as well as cancer progression, has been linked to autophagy dysregulation. Modulating autophagy has been further proposed as a therapeutic treatment for both cancer and cardiovascular disorders. The safe and effective use of autophagy modulation as a cardioprotective strategy during cancer treatment especially requires careful consideration and experimentation to minimize the impact on cancer treatment. We focus here on recent advances in targeted autophagy modulation strategies that utilize interdisciplinary approaches in biomedical sciences and are potentially translatable to treat cardiotoxicity and improve cancer treatment outcomes. This review highlights non-small molecule autophagy modulators to enhance targeted therapy, nanomedicine for autophagy modulation and monitoring, and in vitro models and future experiments needed to bring novel autophagy discoveries from basic research to clinical translation.

Carvedilol (CAR) is an FDA-approved adrenergic blocker commonly given to cardiovascular disease patients. Interestingly, CAR also shows anti-cancer potential in reducing cancer-specific mortality in breast cancer patients. To advance CAR usage in cardio-oncology, we hypothesize that elucidating the molecular mechanism of the energy sensing and homeostasis pathways underlying CAR’s cardioprotective capabilities can enhance its clinical translation. Canine cardiac slices were generated from 3 euthanized pet dogs free of cardiovascular disease. CAR pretreatment (1μM, 4 hrs) with or without a 24-hr exposure to 5μM of cardiotoxic doxorubicin (DOX) was performed. Murine slices from 5 C57Bl6 mice with the same treatments as above was also performed. Direct tissue imaging on IVIS Spectrum was carried out to assess apoptosis by Annexin V staining, autophagy with autophagy detecting nanoparticle (ADN) developed in-house, and DOX retention in tissue by the inherent DOX fluorescence. Tissue was analyzed by histological staining of apoptosis and Western blot of autophagy and energy sensing pathways. Energetics quantified by Seahorse assay was performed in isolated mitochondria from cardiac slices in rat cardiomyoblasts (H9C2 cells) and human iPSC-induced cardiomyocytes (iCells). In both canine and murine cardiac slices, autophagy was significantly reduced (p<0.05) by DOX while apoptosis was significantly increased (p<0.05), both of which were reversed by CAR. CAR did not change DOX fluorescence in tissue. Western blot of autophagy biomarkers LC3, p62 and Beclin-1 confirmed that autophagy impaired by DOX was restored by CAR. The beneficial autophagy restoration by CAR was modulated by a significantly increased phosphorylation of AMP kinase (AMPK), a key energy sensing pathway that activates autophagy via significantly reducing mTOR (p<0.0001). In mitochondria isolated from cardiac slices, basal respiration and ATP production that were significantly impaired by DOX were rescued (p<0.0001) by CAR. Similar rescue of basal respiration and ATP production by CAR was seen in murine and human cardiomyocytes, suggesting that CAR exerts a direct protective effect on the at-risk cardiomyocytes during DOX stress. We demonstrated for the first time in human, canine, and murine that CAR targets the AMPK pathway to activate autophagy, reduce cardiomyocyte apoptosis, and restore mitochondrial energetics, thus with immense translational potential in cardioprotection.

Autophagy is a key biological process that has proven extremely difficult to detect noninvasively. To address this, an autophagy detecting nanoparticle (ADN) was recently developed, consisting of an iron oxide nanoparticle decorated with cathepsin-cleavable arginine-rich peptides bound to the near-infrared fluorochrome Cy5.5. Activation of the probe in autophagolysosomes results in the emission of Cy5.5 fluorescence and provides a measure of autophagosome flux. However, in the early autophagosome ADN fluorescence is silent due to fluorochrome stacking. Here, we introduce to ADN a second non-cleavable fluorophore that allows the probe to be tracked through all stages of autophagy. The nature of the secondary/tracking fluorophore has a profound effect on the activation of ADN and the emission of Cy5.5 fluorescence. The lead candidate, ADN2 (featuring AZDye546 as the secondary fluorophore) has the highest activation rate and change in Cy5.5 fluorescence. Absorbance and fluorescence spectrophotometry methods show that the negatively charged AZDye546 interacts with the positively charged polyarginine motifs of the Cy5.5-polyArg activatable fluorophore, resulting in enhanced baseline quenching of the Cy5.5 signal in the nanoprobe. Flow cytometry shows that the activation of ADN2 remains specific for autophagy and is strongly modulated by classical regulators of autophagy (starvation, bafilomycin) and genetic deletion of key autophagy proteins (ATG5, ATG7). ADN2 co-localized strongly with LC3-GFP positive autophagosomes and provided readouts of in vivo probe delivery and activation in the hearts of fed/starved mice. ADN2 enhances the ability to image autophagy without genetic transfection of cells/animals and underscores the possible effects for unanticipated interactions between fluorochromes and other moieties on the surface of decorated nanoparticles.

NETosis, the process of neutrophil cell death due to the formation and release of neutrophil extracellular traps (NETs) has been implicated in the pathogenesis of heart disease. NETs are composed of immunogenic DNA fragments and citrullinated histones. The role of NETosis in the context of cardiac arrest and resuscitation (CA/R) is however unknown. We previously reported the development of a Dextran-Thiazole Orange (DTO) nanoprobe, with nanomolar affinity for nucleic acids, that exerts acute anti-inflammatory effects and reduces ischemia-reperfusion injury in mice. We aimed here to determine whether the DTO nanoprobe would also bind to the nucleic acid in NETS, whether DTO could attenuate the intensity of NETosis, and whether this would improve outcomes and survival after cardiac arrest. C57Bl6 mice were arrested for 8 minutes before resuscitation. DTO or unmodified dextran (vehicle control) was injected at the time of resuscitation, and again 4 hours later (n=38 mice). Survival and neurological function were scored daily. Time-domain resolved (lifetime) and conventional fluorescence imaging was performed at 4 hours after resuscitation in 6 additional mice injected with DTO. NETosis was evaluated by protein blot in the hearts of cardiac arrest mice injected with DTO or control. DTO significantly (p<0.05) improved 10-day survival (Fig. A), mean survival (from 4 to 7 days, Fig. B), and neurological function (Fig. C) following CA/R. Lifetime imaging (Fig. D), but not conventional fluorescence imaging (Fig. E) revealed the presence of DTO in the injured heart, liver and kidneys. In the heart, DTO signal was significantly increased in CA/R compared to sham mice injected with DTO or CA/R mice injected with control dextran (Fig. F). Citrullinated H3 and HMGB-1 (High mobility group box 1, a chromatin protein) were both upregulated in CA/R, and were significantly attenuated by DTO injection (Fig. G-I). We show for the first time that NETosis plays a key role in cardiac arrest and resuscitation. DTO is capable of detecting NETosis as well as attenuating it, likely via a reduction in HMGB-1 activity. The modulation of NETosis may provide a new avenue to improve survival after cardiac arrest.

Objective: Cardiotoxicity from cancer therapy is a rising clinical issue due to a lack of cardioprotective strategies that can be safely applied to cancer patients without diminishing chemotherapeutic efficacy. Modulating autophagy, a conserved cellular homeostasis pathway, can be beneficial by reducing apoptosis in heart disease. However, the impact of upregulated autophagy on cancer and cancer therapy remains unknown, and thereby, a major obstacle in clinical translation. We aim to study the pathophysiology of autophagy in the heart and in tumor during cancer therapy. Hypothesis: Using a novel Autophagy Detecting Nanoparticle (ADN) probe developed by our lab for in vitro and in vivo fluorescence quantification of autophagy, we hypothesize that direct imaging of autophagy may predict the differential impact of autophagy modulation on apoptosis in the heart and tumor in murine and human models. Methods: Cardiomyocytes (rat H9C2, human iPSC-CMs) and breast cancer cell lines (mouse 4T1-Luc, human MDA-MB-231 and MCF7) were treated with cardiotoxic doxorubicin (Dox) with or without either autophagy-activating (rapamycin (Rp, 0.1 μM) or starvation (Stv)), or autophagy-inhibiting (chloroquine (Cq, 20 μM)) conditions for 6-96 hours. Cell viability (MTT assay and Real-Time Cell Analysis), apoptosis (Annexin V), and autophagy levels (flow cytometry with ADN) were measured. Tumor formation from implanted 4T1-Luc in BALB/c mice (n=10) was measured, and the impact of acute 24-hour Stv on apoptosis and autophagy in the heart and tumor was simultaneously investigated following Dox (15 mg/kg) treatment. Results: ADN signal showed Dox impaired autophagy in cardiomyocytes and cancer lines 4T1-Luc and MCF7. Autophagy activation significantly reduced Dox-induced apoptosis after 24 hours, improved long-term viability of cardiomyocytes, and did not adversely affect the Dox killing of 4T1-Luc and MCF7 cells. In MDA-MB-231 cells, Dox activated autophagy which remained high when co-treated with Rp, and interestingly was further augmented by Cq, presumably by triggering autosis leading to enhanced Dox efficacy. In tumor bearing mice treated with Dox, Stv restored autophagy levels and reduced apoptosis in the heart after 24 hours, and rescued heart function at 4 weeks. Stv significantly increased autophagy and apoptosis levels in tumors after 24 hours, leading to reduced tumor volume over time and showing no adverse effect of acute Stv on Dox efficacy. Conclusions: ADN imaging of autophagy levels in vitro and in vivo reveals differential heart and tumor responses to autophagy modulation during cardiotoxic cancer therapy, and may further serve as a viable biomarker for evaluating cardioprotective strategies. NIH, American Heart Association This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Introduction: The FHOD3-V1151I variant has been identified by multiple Genome Wide Association Studies to be associated with hypertrophic cardiomyopathy (HCM). FHOD3 regulates actin filament formation which is central to key cardiomyocyte processes including autophagy. The precise role of autophagy in cardiomyopathy is an emerging area of research but poorly characterized. We hypothesize that V1151I may perturb FHOD3-dependent cardiac autophagy regulation. Methods: The homologous V1151I variant was introduced into mice using CRISPR. Cardiac function was studied by echocardiography, cardiac mitochondrial function measured by Seahorse (Agilent) and oil red O staining of lipid droplets in cardiac sections. Cultured H9C2 cardiomyoblasts were transfected with FHOD3-V1151V (WT) or V1151I (variant) expression plasmids and immunoprecipitation and immunofluorescence were performed to assess protein interaction and localization. Phosphatidylinositol 3-phosphate (PI3P, Echelon) was used for the rescue experiments. Results: Heart tissue from FHOD3-V1151I variant mice (n=7) showed a 5-fold (p<0.0001) increase in lipid droplet accumulation and impaired mitochondrial respiration and ATP production (p<0.0001), while cardiac structure and function assessed by echocardiography was similar to V1151V WT littermate control hearts (n=7). A bioinformatics analysis predicted FHOD3 interaction with PFN4 which we confirmed by co-immunoprecipitation in addition to ATG16, a regulator of autophagic vesicle membrane biogenesis. The FHOD3-PFN4-ATG16 interaction was however lost in FHOD3-V1151I hearts. Immunofluorescence of H9C2 cells expressing FHOD3-V1151I revealed that the variant disrupts interaction of LC3 with PI3P, a hallmark of early autophagy. The disrupted LC3-PI3P interaction seen with the V1151I variant phenocopies cells treated with early autophagy inhibitors 3-MA or spautin, but not late autophagy inhibitor chloroquine. Furthermore, exogenous PI3P restored the molecular interaction and autophagy dysregulation. Conclusions: We demonstrate for the first time that early autophagy dysregulation underlies energetic defects observed in cardiomyopathy, and is reversible, thus providing a novel potential therapeutic target.

Background: Obese and pre-diabetic women have a higher risk for cardiovascular death than age-matched men with the same symptoms, and there are no effective treatments. We reported that obese and pre-diabetic female Zucker Diabetic Fatty (ZDF-F) rats recapitulate metabolic and cardiac pathology of young obese and pre-diabetic women and exhibit suppression of cardio-reparative AT2R. Here, we investigated whether NP-6A4, a new AT2R agonist with the FDA designation for pediatric cardiomyopathy, mitigate heart disease in ZDF-F rats by restoring AT2R expression. Methods: ZDF-F rats on a high-fat diet (to induce hyperglycemia) were treated with saline, NP-6A4 (10 mg/kg/day), or NP-6A4 + PD123319 (AT2R-specific antagonist, 5 mg/kg/day) for 4 weeks (n = 21). Cardiac functions, structure, and signaling were assessed by echocardiography, histology, immunohistochemistry, immunoblotting, and cardiac proteome analysis. Results: NP-6A4 treatment attenuated cardiac dysfunction, microvascular damage (−625%) and cardiomyocyte hypertrophy (−263%), and increased capillary density (200%) and AT2R expression (240%) (p < 0.05). NP-6A4 activated a new 8-protein autophagy network and increased autophagy marker LC3-II but suppressed autophagy receptor p62 and autophagy inhibitor Rubicon. Co-treatment with AT2R antagonist PD123319 suppressed NP-6A4’s protective effects, confirming that NP-6A4 acts through AT2R. NP-6A4-AT2R-induced cardioprotection was independent of changes in body weight, hyperglycemia, hyperinsulinemia, or blood pressure. Conclusions: Cardiac autophagy impairment underlies heart disease induced by obesity and pre-diabetes, and there are no drugs to re-activate autophagy. We propose that NP-6A4 can be an effective drug to reactivate cardiac autophagy and treat obesity- and pre-diabetes-induced heart disease, particularly for young and obese women.

Chemotherapy-induced impairment of autophagy is implicated in cardiac toxicity induced by anti-cancer drugs. Imperfect translation from rodent models and lack of in vitro models of toxicity has limited investigation of autophagic flux dysregulation, preventing design of novel cardioprotective strategies based on autophagy control. Development of an adult heart tissue culture technique from a translational model will improve investigation of cardiac toxicity. We aimed to optimize a canine cardiac slice culture system for exploration of cancer therapy impact on intact cardiac tissue, creating a translatable model that maintains autophagy in culture and is amenable to autophagy modulation. Canine cardiac tissue slices (350 μm) were generated from left ventricular free wall collected from euthanized client-owned dogs (n = 7) free of cardiovascular disease at the Foster Hospital for Small Animals at Tufts University. Cell viability and apoptosis were quantified with MTT assay and TUNEL staining. Cardiac slices were challenged with doxorubicin and an autophagy activator (rapamycin) or inhibitor (chloroquine). Autophagic flux components (LC3, p62) were quantified by western blot. Cardiac slices retained high cell viability for >7 days in culture and basal levels of autophagic markers remained unchanged. Doxorubicin treatment resulted in perturbation of the autophagic flux and cell death, while rapamycin co-treatment restored normal autophagic flux and maintained cell survival. We developed an adult canine cardiac slice culture system appropriate for studying the effects of autophagic flux that may be applicable to drug toxicity evaluations.

Background: Obese and pre-diabetic women have a higher risk for cardiovascular death than age-matched men with the same symptoms, and there are no effective treatments. We reported that obese and pre-diabetic female Zucker Diabetic Fatty (ZDF-F) rats recapitulate metabolic and cardiac pathology of young obese and pre-diabetic women and exhibit suppression of cardio-reparative AT2R. Here, we investigated whether NP-6A4, a new AT2R agonist with the FDA designation for pediatric cardiomyopathy, mitigate heart disease in ZDF-F rats by restoring AT2R expression. Methods: ZDF-F rats on a high-fat diet (to induce hyperglycemia) were treated with saline, NP-6A4 (10 mg/kg/day), or NP-6A4 + PD123319 (AT2R-specific antagonist, 5 mg/kg/day) for 4 weeks (n = 21). Cardiac functions, structure, and signaling were assessed by echocardiography, histology, immunohistochemistry, immunoblotting, and cardiac proteome analysis. Results: NP-6A4 treatment attenuated cardiac dysfunction, microvascular damage (-625%) and cardiomyocyte hypertrophy (-263%), and increased capillary density (200%) and AT2R expression (240%) (p < 0.05). NP-6A4 activated a new 8-protein autophagy network and increased autophagy marker LC3-II but suppressed autophagy receptor p62 and autophagy inhibitor Rubicon. Co-treatment with AT2R antagonist PD123319 suppressed NP-6A4's protective effects, confirming that NP-6A4 acts through AT2R. NP-6A4-AT2R-induced cardioprotection was independent of changes in body weight, hyperglycemia, hyperinsulinemia, or blood pressure. Conclusions: Cardiac autophagy impairment underlies heart disease induced by obesity and pre-diabetes, and there are no drugs to re-activate autophagy. We propose that NP-6A4 can be an effective drug to reactivate cardiac autophagy and treat obesity- and pre-diabetes-induced heart disease, particularly for young and obese women.

Autophagy-the lysosomal degradation of cytoplasmic components via their sequestration into double-membraned autophagosomes-has not been detected non-invasively. Here we show that the flux of autophagosomes can be measured via magnetic resonance imaging or serial near-infrared fluorescence imaging of intravenously injected iron oxide nanoparticles decorated with cathepsin-cleavable arginine-rich peptides functionalized with the near-infrared fluorochrome Cy5.5 (the peptides facilitate the uptake of the nanoparticles by early autophagosomes, and are then cleaved by cathepsins in lysosomes). In the heart tissue of live mice, the nanoparticles enabled quantitative measurements of changes in autophagic flux, upregulated genetically, by ischaemia-reperfusion injury or via starvation, or inhibited via the administration of a chemotherapeutic or the antibiotic bafilomycin. In mice receiving doxorubicin, pre-starvation improved cardiac function and overall survival, suggesting that bursts of increased autophagic flux may have cardioprotective effects during chemotherapy. Autophagy-detecting nanoparticle probes may facilitate the further understanding of the roles of autophagy in disease.

Introduction & Hypothesis: Untreated obesity induces insulin resistance and heart disease affecting 42% of the US population. Autophagy, a conserved homeostatic process for cellular quality control, plays a critical role in maintaining health. Heart tissues of obese humans and rodent models show impaired autophagy. Drugs that can regulate autophagy are thus of high clinical significance. We reported recently that NP-6A4, a new peptide agonist of the cardiovascular protective angiotensin receptor AT2R, mitigated obesity-induced cardiac dysfunction and damage in rats with untreated obesity and pre-diabetes. We hypothesized that activation of autophagy by NP-6A4 is involved in NP-6A4-AT2R-induced cardioprotection in untreated obesity. Methods & Results: 7-week old female ZDF rats fed high fat diet (D12468, Research Diet) for 6 weeks were randomized to receive either NP-6A4 (10mg/kg/day), NP6A4+AT2R antagonist PD123319 (PD, 5mg/kg/day) delivered subcutaneously (N=6/group), or vehicle (N=5) for 4 weeks. Echocardiography showed that NP-6A4 reduced abnormal E/A ratio from 2.1 to 1.45 (P<0.006), and isovolumic contraction (P<0.004) and relaxation times (P<0.02), and increased myocardial radial strain (P<0.019) and radial strain rate (P<0.033). PD reversed these effects. Immunohistochemistry (IHC) and Western blot analysis revealed a significant 5.9-fold increase (p<0.0006) in LC3 puncta (Fig. A-B) and 2.2-fold increase (p<0.0008) in lipidated autophagy marker LC3-II (Fig. C) compared to vehicle controls. The autophagy activation by NP-6A4 was not seen with the co-administration of PD (Fig. A-C). Cardiac proteome analysis showed that Sec16a, an important partner of autophagy initiators ULK1/2 is increased in ZDF rat heart by NP-6A4 (2.75 fold; P<0.020), but PD suppressed this effect. Conclusions: We identified NP-6A4’s dual targeting of AT2R and novel autophagy regulation to mitigate cardiac dysfunction induced by untreated obesity in female rats

Background: Cardiotoxicity due to chemotherapy remains a challenge. Cardioprotective interventions have not slowed the progression to heart failure. Cardiomyocyte (CM) death can be attributed to autophagy impairment during chemotherapy-induced toxicity. We hypothesize that autophagy activation can be a viable cardioprotective strategy. Methods: H9C2 rat cardiac myoblasts and cardiac slices generated from euthanized dog hearts were treated with 5-50μM doxorubicin (Dox) +/- autophagy activators for 24 hours. Annexin V (Anx) and TUNEL detection of apoptosis, MTT assay of cell viability, and western blots were performed. C57Bl6 mice were challenged with either single Dox (15 mg/kg i.p.), or repeated doses (4 mg/kg i.p., weekly, 5x) +/- 24-hour pre-starvation. Cardiac function was assessed by echo. Results: Dox significantly increased H9C2 apoptosis, rescued by rapamycin (Fig. A). Autophagy induction by phosphatidylinositol-3-phosphate (Pi3P) restored autophagic flux via downregulating autophagy-inhibitory Rubicon (Fig. B). Autophagy stimulation by Pi3P, statins, or starvation significantly reduced H9C2 apoptosis and improved viability (Fig. C-E), but not in human cancer cells. Single high Dox dose in mice markedly increased CM apoptosis by 24 hours, attenuated by pre-starvation without perturbing Dox retention in the heart (Fig. F). In chronically treated Dox mice, starvation before each Dox dose improved cardiac function and survival (Fig. G-H). Viable dog cardiac slices generated (Fig. I-J) exhibited preserved autophagic flux (Fig. K). Dox (5μM, 30-min exposure) induces autophagy blockade at 24 hours leading to apoptosis (Fig. L). Autophagy restoration by rapamycin reduced apoptosis in cardiac slices but not primary canine cancer cells (Fig. M). Conclusions: Autophagy restoration via modulating Rubicon exerts anti-apoptotic effects in murine and canine CMs, but not in cancer cells, potentially translatable to impact cardio-oncology treatment.

Dogs with cancer treated with chemotherapy agents such as doxorubicin (DOX) develop cardiovascular toxicity, providing an opportunity to evaluate cardioprotective strategies in the setting of cancer treatment translatable to human disease. However, due to the lack of a suitable approach to culture primary adult canine cardiomyocytes, mechanistic interrogation of cardiotoxicity after cancer therapy remains a challenge. Our study thus aims to validate a canine myocardial slice culture model to study autophagy modulation and the role of extracellular vesicle (EV) associated miRNA in the setting of DOX induced cardiotoxicity. We hypothesize that induction of autophagy in canine myocardial tissue will reduce apoptosis, exert early changes to EVs, and ameliorate DOX cardiotoxicity. Left ventricular tissue from client-owned donated adult dog hearts was sectioned with a vibratome and viability of the cultured myocardial slices was evaluated by histology and MTT assay. Apoptosis was quantified by TUNEL, and autophagy by fluorescent LC3 protein puncta. Secreted EVs were isolated from cultured tissue by size exclusion chromatography and characterized by nanoparticle tracking analysis, transmission electron microscopy and immunoblot. Canine myocardial slices are consistently viable for 7 days in culture - cardiomyocyte morphology is maintained with low levels of apoptosis, and baseline autophagy is observed. Induction of autophagy with rapamycin treatment results in reduced apoptosis. Cardiac tissue derived extracellular vesicles showed typical size, morphology and enrichment of proteins including tetraspanin CD9 and will be evaluated for changes to EV miRNA profile with autophagy modulation. The canine myocardial slice model allows, for the first-time, the elucidation of complex cross-talk among apoptosis, autophagy, and EVs with molecular and cellular resolutions. Detecting early changes in canine cardiomyocyte autophagy to halt cardiac damage rather than managing its consequences is a paradigm shift translatable towards preventing chemotherapy associated cardiotoxicity.